lm:,.,;,,, (if Shdlflxll Re,\cl/rch, ",,1. 21, No. :!, (lX) 6'10, 2002.

CYTOGENETIC STUDY OF CONCHAPHILA (: ) AND COMPARATIVE KARYOLOGICAL ANALYSIS WITHIN OSTREINAE

ALEXANDRA LEITÂO,t.z RAQUEL CHAVES,2 SARA SANTOS,2 PIERRE BOUDRY 1 HENRIQUE GUEDES-PINTO,2 AND CATHERINE TIDRIOT-QUIÉVREUX3*' IlAboratoire de Génétique et Pathologie, Station de l'Institut Français pour la Recherche et l'Exploitation de la Mer (IFREMER), B.P. 133, 17390 lA Tremblade, France; 2Departamento de Genética e Biotecnologia, /CETA-UTAD, Vila Real, Portugal; 30bservatoire Océanologique, Université Pierre et Marie Curie, Centre National de la Recherche Scientifique, B.P. 28, 06230 Villefranche-sur-Mer, France

ABSTRACT Chromosome preparations of the Olympia Osl,ea conchaphi/a Carpenter were sludied using wnvenlional Giemsa, silver slaÎning, and C-banding techniques. The karyotype consists of six metacentric (l, 2, 4, 6, 8, and 10) and four submetacentric (3, 5, 7, 9) chromosome pairs. The silver-stained nucleolus organizer regions (Ag-NORs) were terminally located on the short anns of the submetacentric pair 5 (56% of cases) and on the long anns of submelscentric pair 7 (6% of cases). Constitutive heterochromatin was observed as telomeric C-bands on the short ann of the NOR-bearîng chromosome pair 5 and as centromeric blocks of several chromosome pairs. Comparative analysis of panerns of karyotype, Ag-NORs, and C-bands of this and of live other flat 0yslers, Oslrea angasi, O. chi/ensis, 0, dense/amellosa, O. edulis, and O. pue/chana, for which data have been previously published, were perfonned, allowing the inference of cytotaxonomic relationships within Ostreinae.

KEY WORDS: Osl,ea conchaphi/a, cytogenetics, cytolaxonomy, Ostreinae

INTRODUCTION chaphila, native to the western United States and Canada, ranges from the southeast Alaska to Baja Califomia (in tidal channels, Studies on oyster cytogenetics have been performed so far on estuaries, bays, and sounds). Commercially impornnt in the laie 26 species of Ostreacea (see Nakamura 1985, Ieyama 1990, 19th century, this species was cultured in the state of Washington Thiriot-Quiévreux 2002). The first data only concemed chromo­ unùl near-collapse of the industry in the 1950s (Baker 1995). some number and gross morphology (Ahmed & Sparks 1967, ln the present work, the karyotype, nucleolus organizer regions Menzel 1968). Later, morphometric measuremencs of chromo­ (NORs), and constitutive heterochromatin distribution were stud­ somes enabled the comparison among karyotypes at the inten>pe­ ied in Oslrea conchaphila (Carpenter 1857) and a comparison with cific and intraspecific level (e.g., Ladron de Guevara et al. 1996, Li previously published karyological data on the five other flat oyster & Havenhand 1997). During the last decade, the development of species mentioned above was perforrned to analyze cytotaxonomi­ banding techniques has allowed the fine characterization of indi­ cal relationships within Ostreinae. vidual chromosomes (e.g., Leitao et al. 1999a), According to the morphologically based classification of Harry (1985), which is currently used, the family includes MATERIALS AND METHODS three subfamilies, that is, Lophinae, Ostreinae, and Crassostreinae. These are sequential hermaphrodites and contain both Specimens of the Californian Olympia oyster Oslrea con· broadcast spawners (Crassostreinae) and brooden> (Lophinae and chaphila (GO) were imported from the Pacific [nsticute (Olympia, Ostreinae). Recent techniques such as molecular phylogenetic WA). Oysters were strictly confined to the quarantine facilities of analysis provided novel insights into oyster evolution and system­ the IFREMER hatchery of La Tremblade, Charente-Maritime, atics (Littlewood 1994, 10zefowicz & 6 Foighil 1998, 6 Foighil & France, according to international recommendalions. After repro­ Taylor 2000). Karyological analysis among cupped oysters, the duction, the progeny (G 1) used in this experiment was reared in the Crassostreinae (Leitào et al. 1999b), has proven complementary to same quarantine facilities for at least 5 mo before sampling. these approaches and has provided additional evolutionary infer­ Whole juvenile animais (ca. 2.5 cm length) were incubated for ences. 7-9 h in a 0.005% solution of colchicine in seawater. The gills Among the flat brooding oyster species, the Ostreinae, five were then removed by dissection and treated for 30 min in 0.9% species have been previously karyologically investigated: Oslrea sodium citrate in distilled water. The material was fixed in a edulis (Linné) (Thiriot-Quiévreux 1984), 0. denselamellosa (Lis­ freshly prepared mixture of absolute alcohol and acetic acid (3: 1) chke) (lnsua & Thiriot-Quiévreux 1991), O. puelchana (Orbigny) with three changes of 20 min each. Fixed pieces of gill from each (lnsua & Thiriot-Quiévreux 1993), O. chilensis (Philippi) (Ladron individual were dissociated in 50% acetic acid with distilled water de Guevara et al. 1994), and O. angasi (Sowerby) (Li & Haven­ solution. The suspension was dropped onto heated slides at 44oC hand 1997). and air-dried (Thiriot-Quiévreux & Ayraud 1982). The Olympia oyster, O. conchaphila (Carpenter 1857), previ­ For conventional karyotypes, gill preparations were stained ously known as 0. lurida (Carpenler 1864), has bec:n studied by with Giemsa (4%, pH 6.8) for 10 min. The silver-staining melhod Ahmed and Sparks (1967) and Ahmed (1973) using squash tech­ for NORs was perforrned on unstained slide preparations accord­ niques and tentative groupingof chromosomes. OSl'en con- ing ta the procedure of Howell and Black (1980), This method only detects those NORs that were active at the precedent inter­ phase (Miller et al. 1976). Chromosomal Ag-NORs can serve as *Corresponding author. E-mail: [email protected] characten> for inferring phylogenetic relationship (c:.g" Amemiya

685 6R6 Lr:l1AO ET AL.

& Gold, 1990). Constitulive heterochromalin regions (C-bands) the chromosomes of21 weIJ-spread metaphases were paired on the were revealed using the method of Sumner (I972) with the coun­ basis ofchromosome size and centromere position. From these, the terstain propidium iodide. The evolutionary significance of the 10 best spreads were used for chromosome measurements and heterochromatin has previously been discussed in vertebrates (e.g., classification (Table 1). The karyotype (Fig. 1A) consists of ten Hsu & Arrighi 1971, Saffery et al. 1999, Chaves et al. 2000). chromosome pairs. Pairs 1, 2, 4, 6, 8, and 10 were metacentric. Images of Giemsa-stained metaphases and C-banding were ac­ Pairs 3, 5, 7, and 9 were submetacentric. quired with a CCD camera (Axioplan, ZEISS) coupied to a ZEISS The Ag-NORs were examined in another 122 metaphases from Axioplan microscope. Digital images were processed using Adobe 10 animaIs. A variable number of one to three Ag-NOR chromo­ Photoshop 5.0 (Windows) using functions affecting the whole of somes were idenlified (Fig. lB). The NOR site was located ter­ the image only. Microphotographs of Giemsa stained metaphases minaIJy on the short arms of the submetacentric pair 5 and on the and C-banding were taken with a ZEISS Axioplan microscope. long arms of the submetacentric chromosome pair 7. The most Digital images were processed using Adobe photoshop 5.0 (Win­ frequent case (56% of observed silver-stained metaphases) wa~ dows). Microphotographs of suitable NOR-stained metaphases one active silver-stained NOR chromosome in pair 5. The Ag­ were taken with a ZEISS III photomicroscope. NORs located on pair 7 occurred in few cases (6%). After karyotyping, chromosome measurements of 10 suitable Constitutive heterochromatin was observed in 31 karyotypes metaphases were made with a digitizer table (Summa Sketch 10 made from well-spread C-banded metaphases from 13 animais. interfaced with a Macintosh. Data analysis was performed with an Telomeric C-bands were always observed on the short arm of the Excel macro-program. Relative length was expressed as 100 limes NOR-bearing chromosome pair 5. In addition, centromeric blocks the absolute chromosome length (in I1m) divided by the total were also found in chromosome pair 2 in 84% of observed length of the haploid complement. Centromeric index was calcu­ metaphases, pairs l, 4, and 5 in 68%, pairs 6 and 8 in 58% of the lated by dividing 100 times the length of the short arm by the total C-banded karyotypes and in fewer cases in pairs 3, 7, and 9 (35%), chromosome length. The arm ratio was determined (Iength of short and in pair 10 (26%) (Fig. IC). arm divided by length of long arm). Both centromeric index and To compare the karyological data from O. conchaphila and arm ratio are given because each expresses centromere position from the other fi ve fiat oyster species previously studied, ideo­ and allows comparison with other karyological studies. Terminol­ grams (Fig. 2) were constructed from relative length and centro­ ogy relating to centromere position (m: metacentric, sm: submeta­ meric index values of O. conchaphila (see Table 1), O. edu/is centric) follows that of Levan et al. (1964). (after Leitiio 2000, French population of La Tremblade hatchery, To elucidate similarities between Ostreinae species, a hierar­ Charentes Maritimes, France), O. angasi (after Li & Havenhand chical agglomeralive flexible clustering program was used (Lance 1997), O. chilensis (after Ladron de Guevara et al. 1994), O. & Williams 1966). Both NOR and centromeric index information denselamellosa (after Insua & Thiriot-Quiévreux 1991), and O. of O. conchaphi/a and five previously studied Ostreinae species puelchana (after lnsua & Thiriot-Quiévreux 1993). The location of were used to cluster species. The Manhattan metric was used to Ag-NORs was a1so included because chromosomal NOR have discriminate and then to associate individual species. Manhattan been used as characters for inferring hypothesis of cytotaxonomic distance appears appropriate to this kind of combination of quan­ relationships (e.g., Amemiya & Gold 1990, LeitAo et al. 1999 b). titative (centromeric index values) and qualitative (NORs posi­ The comparison of the relative length and centromeric index of tions) data and to measure an association between individual ob­ the 10 chromosomes pairs of the studied species showed that pair jects (species) (Legendre & Legendre 1998). 1 was similar arnong a1l species, pair 2 was also similar except for O. pue/chana, pair 3 and 4 were similar except for O. conchaphi/a, RESULTS but taking into account the close relative length and the standard deviation of pair 3 and 4 of O. conchaphila, they may be inverted. Analysis of 60 mitotic metaphase spreads from 15 individuals Pair 5 was variable among species, pairs 6 and 7 were identical of O. conchaphi/a confirmed the diploid chromosome numher of except for O. dense/ame//osa, but in this case, the pairs 6 and 7 2n = 20, scored by Ahmed and Sparks (1967). For karyotyping, cannot he inverted because of their different relative length and the

TABLE 1. Chromosome measurements and classification in 10 cells of Oslrea cOllchaphiill.

Relative Length Arm Rallo Centromeric Index Chromosome Pair No. Mean sn Mean sn Mean SD Classincatlon 1 12.77 0.99 2.39 0.21 42.12 1.78 m 2 11.60 0.55 2.54 0.18 43.87 1.89 m 3 10.64 0.57 1.26 0.20 27.76 2.86 sm 4 10.54 0.50 2.37 0.27 41.94 2.81 m 5 10.47 0.87 1.72 0.16 34.05 2.28 sm 6 9.88 0.78 2.48 0.25 42.81 2.67 m 7 9.46 0.38 1.26 0.16 27.56 2.59 sm 8 9.30 0.58 2.53 0.39 43.59 3.92 m 9 8.79 0.63 1.52 0.25 31.27 3.37 sm 10 6.56 0.65 2.43 0.26 42.18 2.60 m 4 Relative tength 120 100 80 60 40 20 o edulis 2 01 8 6 ,-----O.den 4 2

4 angasi 2 '------O.chi 0 [ 8 X Il [] 6" 4 X l.------O.con 2 o 14 2 chilensis '---t------O.pue 0 1 1 l 1 i 8 )< 1 J 6 X x 1 4 ~-----O.edu 2 1

conchaphi/a L- O.ang

Figure 3. Hierarchlcal agglomeratJve ftexible c1ustering of Ostrea spp. 6 O. den: Ostrea dense14melûJsa; O. chi: O. chiknsis; O. con: O. con­ 4 clulphi14; O. pue: O. puefcha1UJ; O. edu: O. edulis; O. ang: O. ungasi. 2 including six metacentric and four submetacentric chromosome pairs, and the NOR and C-band distribution differ from the other 4 dense/ame/oss osrreinid species studied. The comparison of the relative length 2 and centromeric ·index of the 10 chromosome pairs of the studied 0 " species shows that, if one postulates that shared chromosome pairs 8 with the same relative length and centromeric index may be con­ ~ sidered as primitive, pairs l, 3, and 4 are primitive and pairs 5, 8, 6 )( Il 1 and 10 the most derived. However, these chromosome homologies 4 1 ~ should be confirmed by other banding techniques. 2 1 [. The comparison of karyotypes and location of Ag-NORs 1 Il among species highlighted first the chromosome similarity be­ 41 tween the European species O. edulis and the Australian and New pue/chans 2 • Zealand species O. angasi, aJready pointed out by Li and Haven­ hand (1 997}. Their karyotypes differ slightly (5m, 5 sm in O. 0 i r edu/is and 5m, 3 sm, 2 st in O. angasi), but the phenomenon of 8 >< >< X X variation in the number of submetacentric and subtelocentric chro­ 6 X mosomes have been reported in French populations (Thiriot­ 4 Quiévreux 1984). More striking is that the most frequent Ag-NOR 2 1 patterns are similar in both species. \ The isolated karyotype of O. pue/chana is remarkable because u 2 3 4 5 6 7 8 9 10 Chromosome pair of the single telocentric chromosome. The occurrence of telocen­ tric chromosomes has been only seen in one other species of Os­ Figure 2. Ideograms of six nat oysters constructed from relative lreidae, Dendrostrea fo/ium (Lophinae) (leyama 1990). Iength and centromerfc index values. Stippled chromosome: metacen­ trfc, white chromosome: submetacentric, striped chromosome: subte· The three other flat oysters bear high karyotype resemblance, locentric, black chromosome: telocentrfc. Clrcles indiCJIte Ag·NORs, that is, seven metacentric and three submetacentric pairs for O. dark clrcles the most frequent case. dense/ame/osa and O. chilensis and six metacentric and four sub- 3 4 5 l a- 6 7 8 9 10 B ,

2 3 4 5

6 7 8 9 10 c

2 3 4 5

6 7 8 9 10

Fïl:ure 1. Karyolypes of Os/t-ra cOfld,aphi/a. A. Con\'enlional GlrRlsa slaluinl:: R. silver-s1Ilinr

C-b:mding of pair 6. Pair!! \\ as variabil' ilmcmg spccics. Pair 9 was mSClISSION id..nlÏ<"al ('~œpl for 0. lI/lRasi anu pair III w:ts \'ari:tblc. A slalislical analysis bascd 011 Ci anu NORs (Fig..3) bigh­ This is lhe tirsl rcport on karyol)'pc afler chromosome mea­ lighll:'U the c1uslcring of O. "dl/lis and O. flllgll.li and of 0. rlell.I"­ surements and NORs and C-ballding panems of lhe Olympia o)'s­ IC/mel/Oc Il and O. rhilt'Il.fi.f with O. rfllJ.-11C1l'ilila rlaœu ncar this 1er. The diploid chromo".lme nllmhcr 2/1 ~~ 20 obscr\'cd is char­ dusler. 0. l'l/dd/CUlCl is scpamled (rom the olher spcci~'s by lhe aClerislic of the gcnu", Ostl't'tl and is cmnmon lhroughoul lhe 0 .. ­ hign~! dissilllilanty. l.leD.cetl lNabmllla 1985. Thjriot-Quiévreu~ 1(02). The kar)'olYpe. CYTOGEi':EI1CS Of OST/Œil CONCNAPIllLA 6H9 metacentric pairs for O. conchaphila. Their NOR chromosomal and Havenhand (1997) have also previously disagreed with Harry location revealed thal lhere is a higher resemblance between the (1985), placing O. angasi as a separate species, very close to O. NOR palterns of O. chi/ensis and O. conchaphila lhan between edulis. lhese two species and O. dense/ame/losa. O. chi/ensis and O. con­ Our results show greater congruence with molecular phyloge­ chaphi/a showed terminally located NORs on the short arms of nelic analyses of the Ostreinae, based on partial milochondrial 16S one chromosome pair and on the long arms of another chromo­ rDNA (Jozefowicz & 6 Foighil 1998) and nuclear 28S rDNA (6 some pair. On the contrary, in O. dense/ame/osa, Ag-NORs were Foighil & Taylor 2000) datasets. This is mosl evident for O. edulis always terminally located on the short arms of chromosome pairs. and O. angasi, where a sister species relationship for these Euro­ Data on constitutive heterochromatin dislribution only con­ pean and Australian fIat oysters is strongly supported by both cerned three species, O. dense/amellosa (lnsua & Thiriot­ karyological and gene tree data. The ostreinid milochondrial gene Quiévreux 1991), O. angasi (Li & Havenhand 1997), and O. con­ trees place the six karyologically-characteri7.ed fIat oysters into chaphi/a (this study). Centromeric C-bands were observed in chro­ two clades: one containing (among other taxa) O. puelchana, O. mosome pairs 3, 6, 8, 9, and 10 in 0. angasi and in pairs 6, 8, 9, conchaphi/a, and O. dense/amellosa, the other composed of O. and lOin O. dense/amellosa. Occasional C-bands were seen on lhe edulis, O. angasi, and 0. chi/ensis. With the exception of posi­ centromere of pairs 4 and 7 in O. angasi and on telomeres of pairs tioning of O. chilensis, which in our study is c10ser to O. dense­ 3,5,6,8,9, and 10 in O. dense/amellosa. A substantial proportion /amel/osa, these results are in broad agreement with the topology of the eukaryote genome consists of conslitutive heterochromatin. generated by our statistical analysis based on Ci and NORs. This genornic fraclion includes, among other repetitive sequences, Ali Ostreinae species are ofthe brooding type with an extended satellite DNA. Sequence analysis of these repeats suggests thal the planktotrophic larval development with the exception of O. chil­ sequences are rapidly evolving, and hence they are valuable as ensis, which shows a greatly abbreviated pelagic phase (Walne evolutionary markers; consequenlly, constitutive heterochromatin 1963). This peculiarity is not reflected at the karyological level. analysis can give insighls about the phylogeny relationships of However, O. puelchana is the only brooding oyster with a distinct related species (Saffery el al. 1999, Chaves et al. 2(00). The ob­ dwarf male and it shows a unique phenomenon of settling the servation in O. dense/amel/osa and O. conchaphila of the simul­ larvae on an expansion of the anterior shell margin (Pascual et al. taneous presence of Ag-NORs and C-bands on telomeric position 1989). These unique morphologic features could be related to the in the sarne chromosome pair, that is, pairs 3 and 8 in O. dense­ karyological isolation of O. pue/chana. /ame/losa and pair 5 in O. conchaphila, rnight corroborate the close karyological relationship between these two species noted ACKNOWLEDGMENT above. The cytotaxonomic relationships pointed out here are incon­ This work was partially supported by a Portuguese grant from gruent with the morphologically based classification of Harry the Ministry of Science and Technology (FCTI): SFRHlBPDI (1985), who stated that O. chiLensis and O. angasi were junior 1582-2000. We are grateful to S. Lapègue and D. Cheney for synonymous of O. puelchana in the subgenus Eostrea of the genus supplying live oysters. The authors thank S. Sabini and S. Heu­ Ostrea and that O. edulis and O. dense/amellosa were included in rtebise for excellent technical assistance, R. Ben Hamadou for the subgenus Ostrea ss. The species O. lurida was considered as a statistical analysis, V. Thirio\ for collaboration in Fig. 2, P. Chang junior synonymous of O. conchaphila in the genus Ostreo/a. Li for English editing, and D. 6 Foighil for constructive comments.

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